The Hedgehog (Hh) signaling molecule is a secreted autoproteolytic protein that activates the Hedgehog protein signaling pathway, a signaling pathway that plays a fundamental role in the morphogenesis of many tissues, in particular in the formation of the entoderm and the embryonal axis, the development of the brain and hair follicules, as well as in cell proliferation, and is probably involved in tissue maintenance and repair in the adult (For a review, see: Ingham et al., Genes Dev., 2001, 15, 3059-3087; Marti et al., Trends Neurosci., 2002, 25, 89-96; Weschler et al., Annu. Rev. Neurosci., 2001, 24, 385-428).
The Hedgehog protein and the associated transduction pathway, initially observed in drosophila, are conserved in vertebrates and invertebrates. A single homologue of Hh is present in Drosophila, while three homologues of Hh: Sonic (Shh), Indian (Ihh) and Desert (Dhh) are present in mammals. Of these three homologues, Shh has been the most widely studied because of its extended expression profile during development. Shh participates in ventralization of the neural tube by specifying the early phenotype of several types of neurons along the ventral median line (spinal cord motor neurons, dopaminergic or cholinergic neurons) and by inducing the generation of oligodendrocyte precursors from the ventral spinal cord. Further, Shh induces survival of gabaergic and dopaminergic neurons, orientates the development of serotoninergic precursors and prevents the death of dopaminergic neurons provoked by the toxin MPP. Finally, it induces proliferation of precursors of granular cells in the early post-natal cerebellum. The other members of the Hedgehog family participate respectively in the development of bone tissue (Ihh), the testicles and the peripheral nerves (Dhh). Furthermore, the results obtained with Shh are also applicable to Dhh and Ihh.
The regulatory role of the Hedgehog protein signaling pathway during embryo development has been widely studied: Hh has been associated with maintenance and repair processes in normal tissue and in the spatiotemporal regulation of proliferation and differentiation, thereby allowing developing tissues to reach their correct size with the appropriate cell types and appropriate degrees of vascularization and innervation. The essential role of the Hh signaling function is demonstrated by the dramatic consequences of defects in this signaling pathway in the human fetus, such as holoprosencephaly observed in Sonic Hedgehog mutants.
More recently, the Shh pathway has been identified in the adult brain where the active amino-terminal form of the molecule is expressed in many regions of the mature nervous system at a higher level than that encountered during the early post-natal period (Traiffort et al., Eur. J. Neurosci., 1999, 11, 3199-3214 and 2001, 14, 839-850). Although the roles of Shh in the adult have not been completely elucidated, it appears that, like other neurotrophic molecules, it is a factor that is capable of promoting survival and maintenance of the phenotype of cells of the nervous system (Reilly et al., Mol. Cell. Neurosci., 2002, 19, 88-96; Charytoniuk et al., Eur. J. Neurosci., 2002, 16, 2351-2357). Under pathological conditions, such as a model for Parkinson's disease or a model for peripheral neuropathy, Shh is capable of preserving the axonal projections of dopaminergic neurons in the striatum or of improving the time required for motor recovery consecutive upon crushing of the sciatic nerve (Tsuboi et al., Exp. Neurol., 2002, 173, 95-104; Pepinski et al., J. Pharm. Sci., 2002, 91, 371-387).
Hh proteins are synthesized in the form of immature precursors of approximately 45 kDa that undergo intramolecular cleavage catalyzed by the C-terminal region of the precursor. Said cleavage produces a 25 kDa C-terminal fragment with no known supplemental function and a 19 kDa amino-terminal fragment (denoted HhNp for N-terminal processed domain) bound at its C-terminal end to a molecule of cholesterol, sufficient for all of the known signaling activities of Hedgehog proteins.
The Hedgehog protein signaling pathway comprises three principal components: the Hh ligand, a transmembrane receptor circuit composed of the Patched (Ptc) negative regulator and the Smoothed (Smo) activator, and a cytoplasmic complex that regulates the transcriptional effectors.
The cellular response to the Hedgehog morphogen is controlled by the expression products of the Patched (Ptc) gene, a tumor suppressor gene, and the Smoothened (Smo) proto-oncogene; however, the exact mechanism for Hedgehog pathway regulation has not been completely elucidated. In mammals, there are two Patched genes coding respectively for Ptc1 and Ptc2, glycoproteins with 12 transmembrane domains, homologs of bacterial transporters. The product of the Smo gene that codes for a protein of the family of receptors coupled to G proteins, has no known endogenous ligand. In the absence of Hedgehog, Ptc will block the constitutive activity of Smo. Binding of Hedgehog to Ptc will lift this inhibition and allow signal transduction via Smo. The mechanism for regulating the activity of Smo by Ptc in mammals could involve a molecule transported by Ptc and interacting with Smo (Taipale et al., Nature, 2002, 418, 892-896). The activation of Gli transcription factors is involved in the cascade of events resulting from the activity of Smo. The type I transmembrane protein, HIP (Hedgehog Intercating Protein) constitutes another receptor for Hedgehog molecules which it binds with an affinity comparable to that of Ptc; HIP has been proposed as a negative pathway regulator (Ingham et al., cited above; Ho et al., Curr. Opin. Neurobiol., 2002, 12, 57-63; Taipale et al., Nature, 2001, 411, 349-354). Furthermore, the products of the dispatched (disp) gene, in particular DispA, may be involved in release and accumulation of Hedgehog proteins in the soluble form in the extracellular medium (Ma et al., Cell, 2002, 111, 63-75).
Dysfunctions of the Shh signaling pathway have been associated with a number of cancers, in particular following characterization of Ptc as a tumor suppressor gene. In fact, inactivating mutations of Ptc are associated with Gorlin Syndrome or basocellular naevomatosis, a dominant autosomal disease characterized by cranofacial and cerebral deformities, but primarily by a raised incidence of various tumors, more particularly basocellular carcinomas as regards the skin and medulloblastomas as regards the brain. Mice heterozygous for the Ptc gene develop tumors of the cerebellum, suggesting that a modification of the Shh pathway is at the origin of such tumors (Goodrich et al., Science, 1997, 277, 1109-1113).
Mutations of human Ptc or Smo genes are also observed in primitive neuroectodermal tumors of the central nervous system, principally medulloblastomas (30% of cases), but also in sporadic forms of basocellular carcinomas (40% and 20%, respectively for Ptc and Smo). Furthermore, mutations in Shh (H133Y) are also associated with basocellular carcinomas. Smo mutations, which principally concern two amino acids located in the seventh hydrophobic domain of the receptor (W535L and S533N), induce constitutive activation of the pathway which escapes the negative control of Ptc. In contrast, mutations of Ptc result in a reduction in the inhibition exerted thereby on Smo in the absence of Shh. In both cases, activation of the Shh pathway is the result, leading to a powerful mitogenic activity demonstrated in cultures of precursors of granular cells of the developing brain and to a blockage of the terminal step of differentiation of those neuroblasts (Traiffort et al., Eur. J; Neurosci., 1999, cited above; Charytoniuk et al., J. Physiol. Paris, 2002, 96, 9-16; Dahmane et al., Development, 1999, 126, 3089-3100; Wallace et al., Curr. Biol., 1999, 22, 103-114; Weshler-Reya et al., Neuron., 1999, 22, 103-114). Similarly, the expression of Smo carrying one of these mutations in transgenic mice results in the presence of basocellular carcinomas, demonstrating the direct implication of Smo in the development of these tumors (Xie et al., Nature, 1998, 391, 90-92).
Apart from basocellular carcinomas and medulloblastomas, other types of tumor have been associated with a defect in the Hedgehog signaling pathway; the localization of these tumors is closely correlated with the expression sites of the components of the pathway during embryonic development. Non-limiting examples that may be cited are: breast and meningiomal cancers associated with Ptc mutations, glioblastomas associated with Gli mutations, gastro-intestinal cancers, in particular primary cancers of the stomach, prostate cancers, fibromas and ovarian dermoids, rhabdomyosarcomas, small cell lung cancers, and oral squamous cell carcinomas. Recently, Shh has been associated with psoriasis.
Because of the crucial role played by the Hedgehog protein signaling pathway in many physiological processes and as a consequence of the significance of diseases linked to its dysfunction, the components of this pathway such as the Smoothened, Patched (Patched 1 and Patched 2) proteins, the Dispatched (Dispatched 1 and Dispatched 2) proteins or the HIP protein, represent targets for developing novel molecules that are capable of modulating (activating or inhibiting) this pathway and thus of positively or negatively regulating the development [proliferation, differentiation, migration, survival (apoptosis)] and/or the activity of differentiated and stem cells, in vitro and/or in vivo in the embryo or in the adult.
Such molecules can be used in the treatment of tumors associated with hyperactivation of the Hedgehog pathway: nerve tissue tumors (medulloblastomas, primitive neuroectodermal tumors, glioblastomas, meningiomas and oligodendrogliomas), cutaneous tumors (basocellular carcinomas, trichoepitheliomas), bone and muscle tissue tumors (rhabdomyosarcomas, osteosarcomas) and tumors of other tissues (kidney, bladder).
Such molecules can also be used in the treatment of neurodegenerative type diseases necessitating blockage of the Hedgehog pathway (Parkinson's disease, Huntington's chorea, Alzheimer's disease, multiple sclerosis, motor neuron disease), and diseases in which a blockage of the Hedgehog signaling pathway could be beneficial, such as diabetes.
Such molecules are also useful in the medical or surgical treatment (plastic or reparative surgery, tissue or organ transplants) of numerous acute, sub-acute or chronic, genetic or acquired pathologies—involving a tissue dysfunction linked to deregulation of the Hedgehog pathway—to induce the formation, regeneration, repair and/or to increase the activity of tissues; non-limiting examples are: nerve tissue [central nervous system (brain) and peripheral nervous system (sensory, motor, sympathetic neurons)], bone, cartilage, testicles, liver, spleen, intestine, pancreas, kidneys, smooth and skeletal muscles, the heart, lungs, skin and hair system, the mucous membranes, blood cells and cells of the immune system. Non-limiting examples of such pathologies that may in particular be cited are neuropathies and associated neuromuscular diseases, diabetes, alopecia, burns, ulcers (skin and mucous membranes) and problems with spermatogenesis.
Various molecules that are capable of modulating the activity of the Hedgehog pathway have been identified:                Hedgehog proteins and derivative polypeptides (fragments, variants . . . ), in particular Hedgehog proteins antagonists (International application PCT WO 01/98344 in the name of BIOGEN); because of their size, such proteins and polypeptide derivatives cannot pass through the hematoencephalic barrier and thus cannot be administered systemically, in particular for the treatment of cerebral tumors linked to hyperactivation of the Hedgehog protein signaling pathway. Furthermore, such molecules are difficult to produce and to purify and have poor stability;        heterocyclic organic molecules (International application PCT WO 01/74344 in the name of CURIS and Chen et al., PNAS, 2002, 99, 14071-14076);        nitrogen-containing heterocyclic molecules (International applications PCT WO 01/19800, WO 01/26644 and WO 02/30421 in the name of CURIS and Kamenetsky et al., J. Biol., 2002, 1, 1-19); and        plant steroids derived from Veratrum spp (jervine, cyclopamine and cycloposine) and from Solanum spp. (solanidine), substituted in position 16, 17 or 18 with an amine or an amine derivative, and cholesterol: American patent U.S. Pat. No. 6,432,970 and International applications PCT WO 99/52534 and WO 01/27135 in the name of JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE; American patent U.S. Pat. No. 6, 291,516 in the name of CURIS; International application PCT WO 00/41545 in the name of ONTOGENY; International application PCT WO 02/30462 in the name of CURIS; Talpale et al., Nature, 2000, 406, 1005-1009; Berman et al., Science, 2002, 297, 1559-1561. However, cyclopamine is a teratogenic agent at the origin of holoprosencephaly and cyclopia in the embryo in mammals and the absence of toxicity to mammals of other compounds derived from plant steroids has not yet been demonstrated;        mifepristone (17β-hydroxy 11β-(4-dimethylamino phenyl) 17α-(prop-1-ynyl)estra-4,9-dien-3-one), also denoted RU-486 or RU-38486 (French patent FR 03 00646 in the name of CNRS), for which an inhibiting activity of the activity of the Hedgehog protein signaling pathway has been demonstrated.        
It appears from the foregoing that there is currently no effective molecule for the treatment of pathologies necessitating an inhibition of the activity of the Hedgehog protein signaling pathway for which an absence of toxicity has been established by clinical trials in man.
As a consequence, the inventors set themselves the target of providing novel compounds that are inhibitors of the Hedgehog protein signaling pathway that are more practical, and are in particular simple to synthesize and potentially useful in human therapy.